Corneal therapeutic agent

ABSTRACT

A corneal therapeutic agent comprises at least one of a hexapeptide and a pharmacologically acceptable salt thereof, represented by formula (I) shown below, and a pharmacologically acceptable carrier, 
     
         A-B-Pro-C-D-E                                              (I) 
    
     where A is L- or D-arginine or lysine whose N-terminal amino group is deaminated, alkylated or acylated; B is L- or D-arginine, lysine or hystidine; Pro is L- or D-proline; C is L- or D-tyrosine, tryptophane, or phenylalanine; D is L- or D-valine, isoleucine, or leucine having an amino group whose hydrogen atom may be substituted with an alkyl group having 1 to 4 carbon atoms; E is L- or D-valine, isoleucine or leucine having a C-terminal carboxylic group which is unsubstituted or substituted with --COOR, --CH 2  OR or --CONHR, where R represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

This application is the U.S. National stage of PCT/JP95/01549 filed Aug.4, 1995.

TECHNICAL FIELD

The present invention relates to a corneal therapeutic agent, and moreparticularly, to a corneal therapeutic agent which accelerates thehealing of the cornea after photon rendering keratectomy using anultraviolet laser, especially, an excimer laser, and which preventscorneal opacity.

BACKGROUND ART

In the ophthalmic field, it has been recently studied to cure paropsiaby geometrical incision of an eye. The corneal incision is employed tocure, for example, ametropia such as myopia, hyperopia, and astigmatismand corneal disorders such as corneal opacity. The surgical treatmentmentioned above is quite advantageous in respect that the permanentcorrection can be realized, compared to conventional eyesight-correctionmethods using compensating lenses such as glasses or contact lenses.

As a representative example of the surgical treatment for paropsia,there is a myopia therapy by means of keratectomy. In particular, radialkeratotomy, one of the surgical treatment methods for ametropia, is usedfor correcting myopia caused by excessive corneal arcuation. In thismethod, cutting is made along the radial lines extending outwardly froma center of the cornea, usually with a surgical knife. The depth of thecutting is generally about 90 to 95% of the cornea thickness. The numberof the cutting lines is possibly in the range of 4-16, generally, 8-12.The corneal incision mentioned above makes the cornea relaxed andslightly flattened, thereby mitigating or overcoming the myopia.

Besides this, photon rendering keratectomy (PRK) using an excimer laseris known. In the PRK method, the central portion of a corneal front faceis cut off to form a depressed portion. The cornea is cut off in theform of a meniscus. In an infrared laser such as a carbonate gas laseror a YAG laser, molecules constituting an irradiated object absorb alaser beam, causing molecular vibration. Heat generated by the molecularvibration melts and cuts the object. In the excimer laser, however,photon energy cuts the bonds of the molecules constituting theirradiated object. As a result, the cornea is rarely denatured withheat. In addition, the laser can process the object precisely, so thatonly required region of the cornea can be cut off accurately to anecessary depth.

However, if the cornea is not completely cured after any surgery, thephenomena called white corneal opacity, namely keratoleukoma, will occurin some cases. The keratoleukoma is caused by random secretion ofcollagen to the corneal surface during a wound-healing stage. The randomcollagen secretion is induced by the stimulation of the cornea with anexcimer laser or the like during a surgical operation.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a cornealtherapeutic agent capable of preventing keratoleukoma by acceleratingthe curing of a cornea after the surgical operation of ametropia.

The present invention provides a corneal therapeutic agent comprising atleast one of a hexapeptide and a pharmacologically acceptable saltthereof (hereinafter, referred to as "HP") represented by formula (I)shown below, and a pharmacologically acceptable carrier,

    A-B-Pro-C-D-E                                              (I)

where A is L- or D-arginine or lysine whose N-terminal amino group isdeaminated, alkylated or acylated; B is L- or D-arginine, lysine orhystidine; Pro is L- or D-proline; C is L- or D-tyrosine, tryptophane,or phenylalanine; D is L- or D-valine, isoleucine, or leucine having anamino group whose hydrogen atom may be substituted with an alkyl grouphaving 1 to 4 carbon atoms; E is L- or D-valine, isoleucine or leucinehaving a C-terminal carboxylic group which is unsubstituted orsubstituted with --COOR, --CH₂ OR or --CONHR, where R represents ahydrogen atom or an alkyl group having 1 to 4 carbon atoms.

The corneal therapeutic agent is developed for curing a corneal woundand preventing corneal opacity, i.e., keratoleukoma, and is used inthese uses.

The corneal therapeutic agent may further contain at least one of anepithelial cell growth factor (hereinafter, referred to as "EGF") andfibronectin (hereinafter, referred to as "FN"), as an active ingredient.In this case, a more excellent effect can be expected.

It is another object of the present invention to provide a cornealtherapeutic kit comprising Agent A containing HP and Agent B containingat least one of EGF and FN. The agent A and the agent B may beadministered separately or simultaneously.

It is still another object of the present invention to provide a cornealtherapeutic kit comprising Agent A containing HP, Agent B containingEGF, and Agent C containing FN. The Agents A, B, and C may beadministered separately or simultaneously.

BEST MODE OF CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in detail.

Amino acids described in the specification will be abbreviated accordingto the IUPAC-IUB biochemical nomenclature (CBN). The amino acids areexpressed as follows:

Arg: L-arginine

Ile: L-isoleucine

Lys: L-lysine

Pro: L-proline

Trp: L-tryptophan

D-Arg: D-arginine

D-Ile: D-isoleucine

D-Lys: D-lysine

D-Pro: D-proline

D-Trp: D-tryptophan

His: L-histidine

Leu: L-leucine

Phe: phenylalanine

Tyr: L-tyrosine

Val: L-valine

D-His: D-histidine

D-Leu: D-leucine

D-Phe: D-phenylalanine

D-Tyr: D-tyrosine

D-Val: D-valine

As six amino acids constituting HP, either D- or L-amino acids may beused.

The active agent HP of the corneal therapeutic agent of the presentinvention has been already proposed as a novel hexapeptide by theapplicants of the present invention (Jpn. Pat. Appln. KOKAI PublicationNo. 5-194590).

In formula (I), the first amino acid A of the N-terminal of thehexapeptide is arginine or lysine whose N-terminal amino group isdeaminated, alkylated, or acylated.

The deamination of the N-terminal amino group, for example, arginine, isachieved by dissolving 5-amino valeric acid in 2N aqueous sodiumhydroxide solution and then adding S-methylthiocarbamide thereto.

Examples of the alkyl group introduced into the N-terminal amino groupof amino acid A are methyl, ethyl, propyl, and butyl. The alkylation ofthe N-terminal amino group can be carried out by reacting amino acid Awith the corresponding alkylbromide, such as methyl bromide or ethylbromide, in an organic solvent such as methylene chloride or pyridine.

Examples of the acyl group introduced into the N-terminal amino group ofamino acid A are formyl, acetyl, propionyl, benzoyl, andp-toluenesulfonyl.

The acylation of the N-terminal amino group can be performed by reactingamino acid A with an acid anhydride such as acetic anhydride, or an acidchloride such as acetyl chloride, in an organic solvent such asmethylene chloride or pyridine.

In formula (I), the peptide bond between fourth amino acid C and fifthamino acid D from the N-terminal of the hexapeptide is represented byformula (II) shown below.

    --CO--NX--                                                 (II)

where X is hydrogen atom or a C₁ to C₄ - alkyl group.

In formula (II), the C₁ to C₄ - alkyl group is, for example, methyl,ethyl, n-propyl, t-propyl, n-butyl, i-butyl, or t-butyl.

In formula (I), amino acid E, the sixth amino acid from the N-terminalof the hexapeptide, is valine, leucine or isoleucine. The C-terminalcarboxyl group of sixth amino acid E may be substituted or unsubstitutedwith --COOR, --CH₂ OR, or --CONHR, where R is a hydrogen atom or analkyl group having 1 to 4 carbon atoms. Examples of the alkyl group aremethyl, ethyl, n-propyl, t-propyl, n-butyl, i-butyl, or t-butyl.

More specific examples of the aforementioned HP following hexapeptidesNo. 1 to 12.

    ______________________________________                                        Peptide No.                                                                            Sequence                                                             ______________________________________                                        1        Desamino-Arg--Arg--Pro--Tyr--Ile--Leu--OH                            2        Desamino-Arg--Arg--Pro--Tyr--Ile-Leucinol                            3        Desamino-Arg--Lys--Pro--Tyr--Ile--Leu--NH.sub.2                      4        Desamino-Arg--Arg--Pro--Trp--Ile--Leu--OEt                           5        N--acetyl--Arg--Arg--Pro--Tyr--Ile--Leu--OH                          6        N--acetyl--Arg--Arg--Pro--Tyr--Ile-Leucinol                          7        N--acetyl--Arg--Lys--Pro--Tyr--Ile--Leu--NH.sub.2                    8        N--actyl--Arg--Arg--Pro--Trp--Ile--Leu--OEt                          9        N--butyl--Arg--Arg--Pro--Tyr--Ile--Leu--OH                           10       N--butyl--Arg--Arg--Pro--Tyr--Ile-Leucinol                           11       N--butyl--Arg--Lys--Pro--Tyr--Ile--Leu--NH.sub.2                     12       N--butyl--Arg--Arg--Pro--Trp--Ile--Leu--OEt                          ______________________________________                                    

The aforementioned HP can be synthesized by either aconventionally-known liquid-phase synthesizing method or a solid-phasesynthesizing method which is generally employed for peptide synthesis.The details of the solid-phase HP synthesizing method are described inJpn. Pat. Appln. KOKAI Publication No. 5-194590.

The active agent of the corneal therapeutic agent of the presentinvention also includes salts of HP. Examples of pharmacologicallyacceptable nonpoisonous salts include a salt of HP with an alkalinemetal such as sodium or potassium; an alkaline earth metal such ascalcium or magnesium; an acid-added salt such as a salt of an inorganicacid including hydrochloric acid, sulfuric acid, phosphoric acid, orcarbonic acid; and an acid-added salt such as a salt of an organic acidincluding acetic acid, propionic acid, tartaric acid, succinic acid,malic acid, aspartic acid, or glutamic acid.

The corneal therapeutic agent of the present invention may be used asophthalmic local preparations. Examples of the ophthalmic localpreparations are eye perfusion liquids, eye drops, eye ointments and thelike. The corneal therapeutic agent of the present invention for use inthe eye perfusion liquid can be prepared by dissolving HP in sterilizedand purified water. In order to make the composition of the eyeperfusion liquid closer to that of aqueous humor, pharmacologicallyacceptable additives such as an isotonizing agent and a buffer can beadded as necessary. Examples of the additives include glucose, sodiumchloride, potassium chloride, calcium chloride, magnesium sulfate,sodium bicarbonate, glutathion, and the like.

The corneal therapeutic agents of the present invention used as an eyedrop include an aqueous eye drop, a non-aqueous eye drop, an opthalmicsuspension, and an opthalmic emulsion. The eye drop is prepared bydissolving or suspending HP in an aqueous solvent such as sterilized andpurified water or saline; or in a non-aqueous solvent, for example, avegetable oil such as cotton seed oil, soybean oil, sesame oil, orpeanut oil. In this case, pharmacologically acceptable additives such asan isotonizing agent, pH controlling agent, viscosity improver,suspending agent, emulsifying agent, and preservative may be added asnecessary. Specific examples of the isotonizing agent includes sodiumchloride, boric acid, sodium nitride, potassium nitride, D-mannitol,glucose, and the like. Examples of the pH controlling agent includeboric acid, sodium sulfite anhydride, hydrochloric acid, citric acid,sodium citrate, acetic acid, potassium acetate, sodium carbonate, borax,and the like. Examples of the viscosity improver include methylcellulose, hydroxypropyl cellulose, polyvinyl alcohol, sodiumchondroitin sulfate, polyvinylpyrrolidone, and the like. Examples of thesuspending agent are polysorbate 80, polyoxyethylene hardened castor oil60, polyoxy hardened castor oil, and the like. Examples of theemulsifying agent are egg-yolk lecithin, polysorbate 80, and the like.Examples of the preservative include benzalkonium chloride, benzetoniumchloride, chlorobutanole, phenylethyl alcohol, paraoxy benzoate, and thelike.

The corneal therapeutic agent of the present invention for the eye dropcontains HP in the range of 10 ng/ml to 100 μg/ml. The eye drop isadministered 1 to 6 times per day and applied 1 to 3 droplets per time.

An eye ointment is also included in the corneal therapeutic agent of thepresent invention. The corneal therapeutic agent of the presentinvention used as the eye ointment can be appropriately prepared byemploying a base material generally used in an eye ointment.

The corneal therapeutic agent for the eye ointment contains HP in therange of 10 ng/ml to 100 μg/ml. The eye ointment may be applied to therear side of an eye lid by means of an eye-drop stick 1 to 6 times perday and 100 μg per time.

The corneal therapeutic agent of the present invention can be applied byattaching it onto contact lenses. In this case, the holding period ofthe agent is increased, improving the efficacy thereof.

The corneal therapeutic agent of the present invention mentioned above,accelerates healing of a corneal wound given bye, e.g., an ametropiaoperation and shortens the time required for the recovery of the corneaafter the operation by virtue of the corneal wound-healing acceleratingeffect. Furthermore, the corneal therapeutic agent of the presentinvention has a corneal-opacity preventing effect, so that cornealopacity, i.e., keratoleukoma, can be cured and prevented.

In the radial keratotomy, the cornea is cut with a surgical knife toform a dissected portion having a V-shape cross section extending fromthe epithelium to the parenchyma via the Bowman's membrane, viewing fromthe surface side of the cornea. It takes several days to cure thedissection after the surgery. In the initial stage, the epithelial cellsare grown along the surface of the dissected wall downwardly from theepithelium, covering the entire wall surface of the dissected portion.Then, the parenchymatous tissue is regenerated at the bottom of thedissected portion. As the parenchymatous tissue is healed from thebottom of the dissected portion upwardly as mentioned above, theepithelial cells, which have been formed on the wall of the dissectedportion in the initial stage, are gradually protruded upward.

However, the portion which has been formed of the parenchymatous tissuebefore the surgical operation is not regenerated completely only by theparenchymatous tissue and partly recouped by the epithelial cells formedat the initial stage of the healing step. Therefore, the cornea afterthe surgical operation is not completely recovered and does not regainthe normal condition. Since the cornea is not completely healed, thecornea is mechanically weakened, generating keratoleukoma in some cases.

In contrast, in the PRK method using an excimer laser, the cornealepithelium is cut off with a surgical knife, and thereafter, the cornealparenchymatous layer is cut off by an excimer laser. Hence, only thesurface portion of the parenchymatous tissue can be cut off. In thismanner, the wound due to the surgical operation can be healed only inthe initial epithelial cell formation stage. As a result, the portioncut off by the excimer laser in the surgical operation is covered by theepithelial cells, thereby regaining almost the normal and healthycondition. However, the epithelial cells of the healed cornea aresometimes nonuniformly formed, so that the surface of the cornealepithelium becomes rough. In addition, when the recovery of theepithelial cells is delayed, keratoleukoma may be developed even afterthe PRK surgery, similarly to the case of the radial keratotomy.

In the foregoing, the PRK method using an excimer laser has beenexplained. In the PRK method, an ultraviolet laser other than an excimerlaser can be used. The ultraviolet laser is referred to a laser having awavelength within the ultraviolet range of about 1 to 400 nm.

When the corneal therapeutic agent of the present invention is appliedto the cornea after the radial keratotomy or the PRK surgery, thehealing of the surgical wound can be accelerated while inflammation ofthe surgical dissection and the cut-off portion and the secretion ofcollagen are being suppressed. Therefore, keratoleukoma is prevented.

As the active gradient of the corneal therapeutic agent of the presentinvention, HP may be used alone or in combination with aconventionally-known compound having a corneal therapeutic activity. Forexample, the corneal therapeutic agent of the present invention maycontain an epithelium growth factor (hereinafter, referred to as "EGF")which is disclosed in Jpn. Pat. Appln. KOKAI Publication Nos. 63-5745and 59-65020, and may contain fibronectin described in Jpn. Pat. Appln.KOKAI Publication No. 63-5745. In this case, more excellent cornealhealing effect can be attained.

Alternatively, both Agent A containing HP and Agent B containing eitherEGF or FN are prepared and administered separately or simultaneously.Agents A and B, each containing an active agent are applied, for examplein the form of ophthalmic local preparations such as an eye drop or aneye ointment. Agents A and B may be administered separately orsimultaneously. The effective amount of EGF used in an eye drop fallswithin the range of 0.01 to 50 μg/ml. The effective amount of FN used inan eye drop falls within the range of 10 ng/ml to 1 mg/ml.

Similarly, Agent A containing HP, Agent B containing EGF, and Agent Ccontaining FN are prepared and also administered separately orsimultaneously.

When HP, EGF, and FN are used together as mentioned above, the initialstage of the corneal healing is accelerated by EGF having an epithelialregeneration growth activity. In addition, FN has a activity concerningan intercellular adhesiveness. Hence, the cornea healing activity of HPcan be accelerated by EGF and FN. As a result, keratoleukoma can beprevented and the cornea can be recovered quickly.

EXAMPLES

Hereinbelow, examples of the present invention will be described indetail.

The following tests were performed with respect to the acceleratingactivity for the corneal wound healing after excimer laser treatment.

1. Sample Preparation

The following substances were used in this test.

HP 1: N-acetyl-Arg-Arg-Pro-Tyr-Ile-Leucinol

HP 2: N-acetyl-Arg-Arg-Pro-Tyr-Ile-Leu-OH

EGF: manufactured by Bio Medical Technologies

FN: manufactured by Bio Medical Technologies

HP 1 and HP 2 were synthesized in accordance with Examples 31 and 32described in Jpn. Pat. Appln. KOKAI Publication No. 5-194590.

HP 1 and HP 2 were diluted with 1.2 ml of an oxyglutathioneye-perfusion/washing solution (trade name: BSS plus, manufactured bySanten Seiyaku) to obtain a diluted solution in a final concentration of0.1 mg/ml. 1000 μg/ml of EGF was diluted with 1 ml of the oxyglutathioneye-perfusion/washing solution to obtain a diluted solution in a finalconcentration of 0.1 mg/ml. FN was diluted with 10 ml of theoxyglutathion eye-perfusion/washing solution to obtain a dilutedsolution in a final concentration of 0.1 mg/ml.

2. Corneal Incision of Test Animal

A colored rabbit was used as a test animal. The following surgicaloperation was applied to the rabbit.

An excimer laser was radiated to the corneas of both eyes of the rabbitunder the condition of 180 mj/10 nsec (10 Hz) per pulse. By thisradiation, the surface of central corneal parenchyma was cut off byabrasion in the form of a meniscus having a diameter of 4.5 mm (9.9diopter) and a maximum center-depth of 70 μm. The excimer laser wasradiated by using Omunimed (trade name) manufactured by Summit (U.S.A.).

3. Sample Administration

Each of the aforementioned samples was administered to both eyes twice aday. A single droplet (about 50 μl) was administrated once in themorning and the afternoon for 5 to 7 days.

                  TABLE 1                                                         ______________________________________                                                           Conc.    Number  Number of                                 Example Sample     (μg/ml)                                                                             of animals                                                                            animal eyes                               ______________________________________                                        control no treatment    1         2                                           1       HP 1       100      1       2                                         2       HP 2       100      1       2                                         3       FN + HP1   100      1       2                                         4       FN + HP2   100      1       2                                         5       EGF + HP1  100      3       6                                         6       EGF + FN   100      3       6                                                 HP1                                                                   7       HP1 + HP2  100      1       2                                                 FN + EGF                                                              ______________________________________                                    

As Comparative examples, eye drops were prepared by dissolving theaforementioned samples in the oxyglutathion eye perfusion/washingsolution in accordance with Table 2 and administered to both eyes of therabbit.

As a control, an excimer laser was radiated to the rabbit, andthereafter, no treatment was provided thereto.

                  TABLE 2                                                         ______________________________________                                                            conc.   Number   Number of                                Example   Sample    (μg/ml)                                                                            of animals                                                                             animal eyes                              ______________________________________                                        control   no treatment  1          2                                          1         FN        100     1        2                                        2         EGF       100     1        2                                        3         FN + EGF  100     3        6                                        ______________________________________                                    

After completion of droplet administration, right and left eyes weretaken out from the rabbit and fixed with a glutathion aldehyde/formalinfixing solution. After the fixation, each cornea including a laserirradiated portion and a non-irradiated portion were excised out. Then,paraffin blocks were prepared in accordance with a customary method andthin-film slices of 5 μm thick were prepared by means of a microtome.The obtained corneal slices were stained with H.E. (hematoxylin-eosin)and then subjected to special staining such as PAS or trichrome.Thereafter, the slices were observed with an optical microscope.

The observation results are as follows:

In the control, no corneal epithelium regeneration was observed at thecenter of the excimer-laser irradiated portion.

In the FN group of Comparative Example 1, epithelium regeneration wasobserved in the entire irradiated portion. However, the thickness of theregenerated epithelium was nonuniform and inflammatory changes wereobserved mainly ascribed to inflammatory cellular infiltration. Inaddition, vacuolation and fibroblast growth were relatively significantat a portion immediately under the epithelium.

In the EGF group of Comparative Example 2, epithelium regeneration wasobserved in the entire irradiated portion. The thickness of theregenerated epithelium is nearly uniform. However, vacuolation andfibroblast growth were observed in the tunica propria under theepithelium. Furthermore, inflammatory changes were observed.

In the FN+EGF group of Comparative Example 3, epithelium regenerationwas observed in the entire irradiated portion. However, the thickness ofthe regenerated epithelium was nonuniform. Vacuolation and fibroblastgrowth were observed in the tunica propria under the epithelium. Inaddition, inflammatory changes were observed.

In the HP1 group of Example 1 and the HP2-group of Example 2, theregeneration of the epithelium was observed in the entire irradiatedportion. However, the thickness of the regenerated epithelium wasnonuniform. Vacuolation and fibroblast growth were observed in thetunica propria. However, inflammatory changes were not observed.

In the FN+HP1 group of Example 3, FN+HP2 group of Example 4, EGF+HP1group of Example 5, epithelium regeneration was observed in the entireirradiated portion. However, the thickness of the regenerated epitheliumwas nonuniform. Vacuolation and fibroblast growth were low in the tunicapropria. However, inflammatory changes were not observed.

In the EGF+FN+HP1 group of Example 6, epithelium regeneration wasobserved in the entire irradiated portion. The thickness of theregenerated epithelium was almost uniform. Slight vacuolation wereobserved under the epithelium. Inflammatory changes were not observed.

In the HP1+HP2+FN+EGF group of Example 7, epithelium regeneration wasobserved in the entire irradiated portion. The thickness of theregenerated epithelium was normal. Vacuolation and fibroblast growthwere rarely observed under the epithelium. Inflammatory changes were notobserved.

From the results above, since the cornea was regenerated in the HP1group of Example 1 and HP2 group of Example 2, the samples of Examples 1and 2 were confirmed to have an corneal wound healing acceleratingeffect, as the same as in the FN group of Comparative Example 1 and theEGF group of Comparative Example 2 which are conventionally known as acorneal therapeutic agent. Furthermore, although inflammatory changeswere observed in the FN group of Comparative Example 1 and the EGF groupof Comparative Example 2, no inflammatory changes were observed in theHP1 group of Example 1 and the HP2 group of Example 2. Hence, it wasfound that the occurrence of inflammation can be prevented. As a result,it was demonstrate that HP1 and HP2 can be used as a corneal therapeuticagent although the thickness of the regenerated epithelium isnonuniform.

On the other hand, in the case where HP1 or HP2 is used together with FNor EGF, as are in the cases of the FN+HP1 group of Example 3, FN+HP2group of example 4 and EGF+HP1 group of Example 5, it was found not onlythat the cornea was regenerated but also that vacuolation and fibroblastgrowth can be suppressed. In addition, occurrence of inflammation wasprevented.

In the case HP, FN and EGF were used together, as are in the cases ofthe EGF+FN+HP1 group of Example 6 and the HP1+HP2+FN+EGF group ofExample 7, the corneal regeneration was observed. In addition, thethickness of the epithelium thereof was uniform. It was furtherconfirmed that vacuolation and fibroblast growth were suppressedsignificantly. Furthermore, occurrence of inflammation can be prevented.In particular, the HP1+HP2+FN+EGF group of Example 7, it wasdemonstrated that the regenerated epithelium had a normal thickness,that vacuolation and fibroblast growth were rarely observed, and that aquite excellent corneal therapeutic effect was exhibited. As described,the corneal therapeutic effect can be enhanced by using HP, FN, and EGFtogether. It was successful to bring the state of the cornea after asurgical operation back closer to the state before operation.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgArgProTyrIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /product="Leucinol"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgArgProTyrIleXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgLysProTyrIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgArgProTrpIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgArgProTyrIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /product="Leucinol"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ArgArgProTyrIleXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ArgLysProTyrIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ArgArgProTrpIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ArgArgProTyrIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /product="Leucinol"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ArgArgProTyrIleXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgLysProTyrIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ArgArgProTrpIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="Arg or Lys"                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /product="Arg, Lys or His"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="Tyr, Trp or Phe"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="Val, Ile or Leu"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /product="Val, Ile or Leu"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      XaaXaaProXaaXaaXaa                                                            15                                                                            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We claim:
 1. A method of preventing keratoleukoma comprising applying tothe eye an amount of a hexapeptide effective to prevent keratoleukoma,wherein said hexapeptide is represented by the formula (I):

    A-B-Pro-C-D-E                                              (I)

where A is L- or D-arginine or lysine whose N-terminal amino group isdeaminated, alkylated or acylated; B is L- or D-arginine, lysine orhistidine; Pro is L- or D-proline; C is L- or D-tyrosine, tryptophane,or phenylalanine; D is L- or D-valine, isoleucine, or leucine having anamino group whose hydrogen atom may be substituted with an alkyl grouphaving 1 to 4 carbon atoms; and E is L- or D-valine, isoleucine orleucine having a C-terminal carboxylic group which is unsubstituted orsubstituted with --COOR, --CH₂ OR or --CONHR, where R represents ahydrogen atom or an alkyl group having 1 to 4 carbon atoms.
 2. Themethod according to claim 1, wherein said hexapeptide is in apharmacologically acceptable carrier.
 3. The method according to claim1, wherein said hexapeptide is selected from the group consistingof:Desamino-Arg-Arg-Pro-Tyr-Ile-Leu-OH;Desamino-Arg-Arg-Pro-Tyr-Ile-Leucinol;Desamino-Arg-Lys-Pro-Tyr-Ile-Leu-NH₂ ;Desamino-Arg-Arg-Pro-Trp-Ile-Leu-OEt;N-acetyl-Arg-Arg-Pro-Tyr-Ile-Leu-OH;N-acetyl-Arg-Arg-Pro-Tyr-Ile-Leucinol;N-acetyl-Arg-Lys-Pro-Tyr-Ile-Leu-NH₂ ;N-acetyl-Arg-Arg-Pro-Trp-Ile-Leu-OEt:N-butyl-Arg-Arg-Pro-Tyr-Ile-Leu-OH;N-butyl-Arg-Arg-Pro-Tyr-Ile-Leucinol;N-butyl-Arg-Lys-Pro-Tyr-Ile-Leu-NH₂ ; andN-butyl-Arg-Arg-Pro-Trp-Ile-Leu-OEt.
 4. The method according to claim 2,wherein said hexapeptide is in the form of an eye drop containing saidhexapeptide or its pharmacologically acceptable salt in an amount of 10ng/ml to 100 ng/ml.
 5. The method according to claim 2, wherein saidhexapeptide is in the form of an eye ointment containing saidhexapeptide or its pharmacologically acceptable salt in an amount of 10ng/ml to 100 ng/ml.